Chemistry of the carboxylic acid of dihydrofluorescein in oxidation and its application to fluorogenic ROS sensing.

The conjugation site of dihydrofluorescein (H2F) is important for the rational design of H2F-based reactive oxygen species (ROS) sensors. Despite the prevalence of H2F analogs detecting cellular ROS, the role of the carboxylic acid of H2F in oxidation is still unclear. To get insight into the conjugation site of H2F, we synthesized H2F diacetate (2) and its amide derivative (3). The absorption and emission spectra of deacetylated 2 and 3 in the presence of H2O2/hematin showed that the carboxylic acid of H2F plays a crucial role in the oxidation of H2F. NMR and HPLC analysis of the oxidation product of deacetylated 3 showed a quantitative and fast generation of non-fluorescent spirolactam (F-Lactam). As regards these observations, we untouched the carboxylic acid at the 3rd position and designed an H2F-based ROS sensor (7) that conjugated the lipophilic chain at the 5th position instead. A series of confocal microscopic experiments of 7 demonstrated that 7 prefers the ER location and that ROS are elevated in the cells by ER stress inducers.

Binary Prodrug of Dichloroacetic Acid and Doxorubicin with Enhanced Anticancer Activity.

The inevitable challenge in conventional chemotherapy is to deliver the anticancer drugs to the dense population of tumors cells while minimizing the drug-associated side effects on the normal cells. Cancer cells' preference for glycolysis for energy production is well recognized. Intuitively, taking advantage of such cancer-associated metabolism would be a promising strategy for anticancer drug delivery with minimal side effects. In this investigation, we have designed a binary prodrug PDOX as a sequential drug delivery regimens to realize the combination therapy for cancer. As cancer cells exhibit abrupt metabolism with elevated pyruvate dehydrogenase kinase (PDK) activity, dichloroacetic acid (DCA, a well-known PDK inhibitor) was used in combination with anticancer drug doxorubicin (DOX). The designed molecular prodrug was activated selectively by cancer-associated esterase to deliver DCA and DOX, respectively, and induced synergetic effects. Hence, sequential targeted delivery of molecular prodrug PDOX offers a promising approach to overcome the offside drug toxicity, pharmacokinetics, and biodistribution of individuals and provide an alternative option for cancer treatment.

Self-Calibrating Bipartite Fluorescent Sensor for Nitroreductase Activity and Its Application to Cancer and Hypoxic Cells.

Aromatic nitro compounds are reduced to their corresponding amino derivatives by nitroreductases (NTR), while identification and characterization of the corresponding enzymes in mammalian systems are yet unrevealed. However, mammalian NTR activity has been considered as a favorable target in development of theranostic agents for cancer and hypoxia of solid tumors. Currently, small molecule-based fluorescent probes have emerged as a valuable assay tool for NTR activity. However, there has been a limit to comparing NTR activity between different cells, since most probes have relied on fluorescence changes that are affected by not only enzymatic activity but also nonenzymatic factors. Here, we developed a self-calibrating bipartite fluorescent probe, consisting of NTR-sensitive nitronaphthalimide and nonsensitive coumarin moieties. Thereby, it was possible to compare the relative NTR activity by monitoring fluorescence ratios in noncancerous and some cancerous cells and to demonstrate for certain that the elevated NTR activity is associated with cancer cells and hypoxia states.

Binary Drug Reinforced First Small-Molecule-Based Prodrug for Synergistic Anticancer Effects.

We developed a small-molecule-based binary drug delivery system (BDDS) with two anticancer drugs, SN-38 and 5'-DFUR. The drug release from the prodrug BDDS can be achieved upon its reaction with intracellular H2O2, overexpressed in cancer cells. The efficacy of BDDS was demonstrated by a comparative study along with that of a single drug conjugate (SDDS), bearing SN-38 alone.

Glycyrrhetinic acid as a hepatocyte targeting unit for an anticancer drug delivery system with enhanced cell type selectivity.

Herein, we report the potential of glycyrrhetinic acid (GA) as an active targeting ligand for hepatocellular carcinoma (HCC) for the development of diagnosis/therapy using small-molecule based approaches. Our preliminary results demonstrated that GA-conjugation to diagnostic/therapeutic counterparts significantly enhanced their HCC targeting ability and excellent therapeutic efficacy.

Fluorogenic reaction-based prodrug conjugates as targeted cancer theranostics.

Theranostic systems are receiving ever-increasing attention due to their potential therapeutic utility, imaging enhancement capability, and promise for advancing the field of personalized medicine, particularly as it relates to the diagnosis, staging, and treatment of cancer. In this Tutorial Review, we provide an introduction to the concepts of theranostic drug delivery effected via use of conjugates that are able to target cancer cells selectively, provide cytotoxic chemotherapeutics, and produce readily monitored imaging signals in vitro and in vivo. The underlying design concepts, requiring the synthesis of conjugates composed of imaging reporters, masked chemotherapeutic drugs, cleavable linkers, and cancer targeting ligands, are discussed. Particular emphasis is placed on highlighting the potential benefits of fluorogenic reaction-based targeted systems that are activated for both imaging and therapy by cellular entities, e.g., thiols, reactive oxygen species and enzymes, which are present at relatively elevated levels in tumour environments, physiological characteristics of cancer, e.g., hypoxia and acidic pH. Also discussed are systems activated by an external stimulus, such as light. The work summarized in this Tutorial Review will help define the role fluorogenic reaction-based, cancer-targeting theranostics may have in advancing drug discovery efforts, as well as improving our understanding of cellular uptake and drug release mechanisms.

Targeted tumor detection: guidelines for developing biotinylated diagnostics.

The challenge in achieving precision medicine relies on how to advance and/or enhance new as well as old therapeutic strategies. Here, we highlight the significant role hydrophilicity of biotinylated fluorescent probe's plays on their cellular uptake behaviour.

Flavin-mediated photo-oxidation for the detection of mitochondrial flavins.

We report a new approach for the detection of mitochondrial flavins through photo-oxidation of a probe molecule. Probe 1 showed high brightness (ε × Φf = 6.50 × 103 M-1 cm-1) at long wavelengths (maximum emission wavelength, λmax = 600 nm) upon photo-oxidation, assisted by the strong electron accepting ability of the isoalloxazine moiety in flavins. Probe 1 also exhibited high selectivity for flavins over various biological oxidants, remarkable photo-stability, and mitochondrial localization.

Indomethacin-guided cancer selective prodrug conjugate activated by histone deacetylase and tumour-associated protease.

An indomethacin-guided drug delivery conjugate (IGDDC) has been designed by utilizing cancer associated elevated HDAC and CTSL activities as consecutive demasking ventures for drug activation. IGDDC exhibited preferential uptake by COX-2 positive cells both in vitro and ex vivo highlighting indomethacin's role in designing new cancer-specific drug delivery frameworks.

A far-red, photo- and bio-stable fluorescent marker selective to the endoplasmic reticulum and its application to tunicamycin-treated HeLa cells.

Herein, we developed an ER selective fluorescent ERp that exhibited a sharp fluorescence emission in the far-red region and high photo- and bio-stability in biological milieu. Its emission is insensitive to pH change and localized in the ER of the cells. Furthermore, it successfully demonstrated that the ER membrane is rapidly reorganized in the perinuclear region by an ER stress inducer, tunicamycin.

BODIPY/Nile-Red-Based Efficient FRET Pair: Selective Assay of Endoplasmic Reticulum Membrane Fluidity.

We synthesized a boron-dipyrromethene (BODIPY)/Nile Red hybrid probe capable of selectively recognizing fluidity changes in the endoplasmic reticulum (ER) membrane due to its preferential localization to the ER and strong energy transfer from BODIPY to the Nile Red moiety, emitting only in nonaqueous environments. ER membrane fluidity in HepG2 cells was markedly reduced by a cell model of metabolic syndrome.

HepG2 Cell Resistance against Camptothecin from a Lysosomal Drug Delivery.

A galactose-appended drug delivery system released camptothecin (CPT) to lysosomes of HepG2 hepatoma cells, resulting in the cell resistance to the anticancer drug. We found that the resistance to CPT is caused by alteration of the drug release from the prodrug in lysosomes, emphasizing that the final delivery locations may critically influence drug efficacy.

Small polyanion recognition of a triazolium cyclodextrin click cluster in water.

In order to detect small polyanions (sPAs), which play important roles in many biological systems, a triazolium cyclodextrin click cluster (5, hexakis{6-(3-methyl-4-hydroxymethyl-1H-1,2,3-triazolium-1-yl)-6-deoxy}-α-cyclodextrin iodide) was synthesized and characterized. The competition binding to 5 occupied by 5-carboxyfluorescein of inositol-1,4,5-trisphosphate (IP3), phytic acid, adenosine triphosphate (ATP), ethylenediaminetetraacetic acid (EDTA), glucose, and glucose-6-phosphate was evaluated by UV/vis titration in HEPES (10 mM, pH 7.4) : methanol (1 : 1, v/v). We obtained the binding constants of IP3 and phytic acid to 5 (1.4 × 10(6) and 1.9 × 10(6) M(-1), respectively); however, the binding constants of ATP and EDTA were significantly lower (2.1 × 10(5) and 4.5 × 10(4) M(-1), respectively). Moreover, glucose and glucose-6-phosphate did not show any detectable binding. In addition, the sPA recognition of the triazolium cyclodextrin click cluster in water was confirmed by fluorescence titration.

Biotin-guided anticancer drug delivery with acidity-triggered drug release.

A novel biotin-guided anticancer drug delivery system, prodrug , consisting of biotin, nitrobenzene, and doxorubicin, with acid-triggered drug releasing capability was synthesized. Its cellular uptake and anticancer activity are selective to the HepG2 cell line over the WI-38 cell line, as revealed by fluorescence confocal microscopic experiments and MTT assay.

Recent development of biotin conjugation in biological imaging, sensing, and target delivery.

Despite encouraging results from preliminary studies of anticancer therapies, the lack of tumor specificity remains an important issue in the modern pharmaceutical industry. New findings indicate that biotin or biotin-conjugates could be favorably assimilated by tumor cells that over-express biotin-selective transporters. Furthermore, biotin can form stable complexes with avidin and its bacterial counterpart streptavidin. The strong bridging between avidin and biotin moieties on other molecules is a proven adaptable tool with broad biological applications. Under these circumstances, a biotin moiety is certainly an attractive choice for live-cell imaging, biosensing, and target delivery.

Enhanced NIR radiation-triggered hyperthermia by mitochondrial targeting.

Mitochondria are organelles that are readily susceptible to temperature elevation. We selectively delivered a coumarin-based fluorescent iron oxide nanoparticle, Mito-CIO, to the mitochondria. Upon 740 nm laser irradiation, the intracellular temperature of HeLa cells was elevated by 2.1 °C within 5 min when using Mito-CIO, and the treatment resulted in better hyperthermia and a more elevated cytotoxicity than HeLa cells treated with coumarin iron oxide (CIO), which was missing the mitochondrial targeting unit. We further confirmed these results in a tumor xenograft mouse model. To our knowledge, this is the first report of a near-infrared laser irradiation-induced hyperthermic particle targeted to mitochondria, enhancing the cytotoxicity in cancer cells. Our present work therefore may open a new direction in the development of photothermal therapeutics.

A cysteamine-selective two-photon fluorescent probe for ratiometric bioimaging.

We report a two-photon fluorescent probe for ratiometric imaging of cysteamine in situ. This probe can detect the levels of endogenous cysteamine with statistical significance in live cells and brain hippocampal tissues, revealing that cysteamine is localized mainly in the perikaria of the pyramidal neurons and the granule cells.

A biotin-guided fluorescent-peptide drug delivery system for cancer treatment.

Herein, we present a fluorescent-peptide drug delivery system composed of biotin-naphthalimide-HJ inhibitor peptide2, prodrug 1. Treatment of 1 to biotin receptor-positive HepG2 cells, which are resistant to high concentrations of the HJ inhibitor peptide2, decreased cell viability and increased intracellular fluorescence.

Chemical sensing of neurotransmitters.

In the past few decades, the development of chemosensors for neurotransmitters has emerged as a research area of significant importance, which attracted a tremendous amount of attention due to its high sensitivity and rapid response. This current review focuses on various neurotransmitter detection based on fluorescent or colorimetric spectrophotometry published for the last 12 years, covering biogenic amines (dopamine, epinephrine, norepinephrine, serotonin, histamine and acetylcholine), amino acids (glutamate, aspartate, GABA, glycine and tyrosine), and adenosine.

Organelle-selective fluorescent Cu2+ ion probes: revealing the endoplasmic reticulum as a reservoir for Cu-overloading.

Two novel Cu(2+) sensors, 1 and 2, bearing naphthalimide and a DPA moiety were synthesized to study copper accumulation in organelles by selective Cu(2+) sensing. The ER-selective Cu(2+) sensor 1 that we developed serves as a valuable tool for understanding the subcellular compartmentalization and roles of copper ions in physiology and pathophysiology.